This document describes how to quantify DNA and RNA concentrations using a UV-visible spectrophotometer. It explains that spectrophotometry uses Beer's Law and Lambert's Law to relate absorbance to concentration and path length. The procedure involves preparing samples, taking blank and sample measurements at 260nm, and using the measured absorbances and dilution factors to calculate concentrations according to the Beer-Lambert Law. Purity is also assessed by calculating absorbance ratios.

1. Techniques in Biotechnology…
M.PHOOL BADSHAH

2. Lecture-7
DNA/RNA Quantification by
Spectrophotometer

3. Introduction
• A first step in many molecular biology investigations is
quantifying the quantity of DNA and RNA.
• An approach that is frequently used to measure nucleic acid
concentrations is UV-visible Spectrophotometry.
• Here's a basic rundown of how a spectrophotometer can be
used to measure DNA and RNA:

4. What is Beer’s Law?
• Beer’s law was stated by August Beer which states that
concentration and absorbance are directly proportional to each
other.
What is Lambert Law?
• Johann Heinrich Lambert stated Lambert law. It states that
absorbance and path length are directly proportional.

5. Equipments and Reagents
• Nucleic Acid: DNA or RNA samples in solution are known as
nucleic acid samples.
• UV-Visible Spectrophotometer: A device that detects the
absorbance of light at certain wavelengths.
• Cuvettes or Microvolume Plates: Containers for holding the
sample during measurement.

6. Procedure:
1- Sample Preparation:
 Use a blank solution (buffer only) for baseline correction and make sure
your DNA or RNA sample is dissolved in an appropriate buffer or solvent.
2- Blank Measurement:
 Put a cuvette into the spectrophotometer that is solely filled with buffer.
 Using the blank solution, set the spectrophotometer to zero absorbance.
3- Sample Measurement:
 Pipette a tiny volume of your DNA or RNA sample into a cuvette or
Microvolume plate (usually 1-2 μL for microvolume instruments, 200-1000
μL for normal cuvettes).
 Insert the microvolume plate or cuvette into the spectrophotometer.

7. 4- Wavelength Selection:
 Measure absorbance at 260 nm (A260) for DNA.
 Measure the absorbance of RNA at 260nm.
5- Absorbance Reading:
 After selecting the wavelengths, note the absorbance values.
6- Calculation of Concentration:
 Using the Beer-Lambert Law, determine the concentration of DNA or RNA
based on the absorbance values:
 For DNA: Concentration (ng/μL)=A260×Dilution Factor×50
 For RNA: Concentration (ng/μL)=A260×Dilution Factor×40
 Any dilution or concentration phases in the sample preparation process are
taken into account by the dilution factor.

8. 7- Purity Assessment:
 Assess the purity of DNA or RNA by determining the A260/A280 ratio. A
pure sample usually has an RNA to DNA ratio of about 2.0 and a DNA
ratio of about 1.8.
 Note:
 While the absorbance at 280 nm indicates the presence of proteins, the
absorbance at 260 nm is specific to nucleic acids and is utilized for
measurement.
 Verify the path length of the spectrophotometer, which is typically 1 cm for
cuvettes, and modify computations as necessary.
 Make that the cuvettes or microvolume plates are clean and that the
spectrophotometer is calibrated correctly.

9.  Contaminants may cause RNA samples to absorb more light at 230 nm.
You can measure A230 to determine purity if necessary.
 Microvolume measurements are frequently performed using nanodrop
spectrophotometers, which enable small sample volumes and minimize
sample consumption.